1. Field of the Invention
The present invention relates generally to improved processes for concentrating desired proteins from fluids and more particularly, to processes for obtaining immunoglobulins from fluids by ultrafiltration through metallic membranes.
2. Background
Filtration is a much used technique for separating wanted substances from those which are unwanted. There are two customary ways in which a feed stream meets a filter: dead-end filtration and cross flow filtration. In dead-end filtration, the feed stream flows perpendicular to the filter surface. In cross flow filtration, on the other hand, the feed stream runs parallel to the filter and the filtrate diffuses across it. The filters used during filtration are often classified by retained particle size. For example, membrane microfilters generally retain particles 0.1-10 microns in diameter, ultrafilters generally retain particles and macromolecules of 1,000-500,000 daltons (approximately 0.001-0.02 microns in diameter), and hyperfilters generally retain molecules of 200-300 daltons. In the laboratory, filter selection is usually straight forward, but scale-up to industrial applications often presents numerous difficulties.
In theory, ultrafiltration should permit the selective separation, concentration, and purification of protein components. The product which passes through the membrane is known as the ultrafiltrate or "permeate" and the product which is retained and concentrated is known as the "retentate".
In practice, ultrafiltration does not proceed according to ideal hypotheses. For example, during ultrafiltration of whey, the retentate has a relatively great residual content of fat, bacteria, and mineral elements which complicate the ideal ultrafiltration process. In addition, most current ultrafiltration membranes have variable pore diameters. Their cut-off capacity is not absolutely accurate and does not correspond to an ideal isoporous membrane. Furthermore, the permeate flux (volume of product per unit of filter area per unit of time) of an ultrafiltration membrane is greatly affected by the presence of a polarization layer or by fouling of the membrane.
Polarization layers form in the course of ultrafiltration and modify the transfer of solutes across the membrane, thereby lowering the permeation rate of the apparatus and changing its separation characteristics. Polarization is caused by convection through the membrane. If fluid flows through the membrane faster than the retained material can diffuse back into the bulk fluid, a saturated layer builds up next to the membrane. The layer's depth and its resistance to flow depend on the speed at which the retentate is circulated. The total permeability of the membrane in the course of operation depends on the polarization layer's thickness and also the nature of its components. The resistance due to fouling builds as deposits chemically bind to the membrane. Fouling is quite distinct from polarization, in which the interfering layer is held against the membrane by hydrodynamic (not chemical) forces.
To obtain highly purified concentrates, filtration may be followed or accompanied by diafiltration which consists of washing the protein concentrates and subjecting them to another filtration. For example, during diafiltration a protein concentrate is brought into contact with the ultrafiltration membrane at the same time as the washing solution, e.g., an aqueous solution. Diafiltration reduces the filterable components from the retentate. It may be a batch process (dilutions followed by successive concentrations) or a continuous process (water is added at the same rate as the permeate is eliminated), In general, protein concentrates of enhanced purity may be obtained by diafiltration.
Roger et al., U.S. Pat. No. 4,485,040 relates to a process of obtaining an alpha-lactalbumin enriched product from whey. The disclosed process carries out a first ultrafiltration on unpasteurized raw whey with membranes having a molecular weight cut-off greater than 5,000 Daltons at a pH between 6.3 and 7 and a temperature between 30.degree. C. and 60.degree. C. to retain the whey proteins. The permeate then undergoes a second ultrafiltration (which is preferably diafiltration) with a membrane capable of retaining alpha-lactalbumin, preferably having a cut-off less than 5,000 Daltons, and recovering the retentate of the second ultrafiltration which constitutes the alpha-lactalbumin enriched product sought. Such a process is reported to fractionate alpha-lactalbumin from whey, but it reportedly does so by means of easily-fouled hollow fiber membranes without use of a pH shift.
Kothe et al., U.S. Pat. No. 4,644,056 relates to a method of preparing a solution of lactic or colostric immunoglobulins or both. In the disclosed method, milk or colostrum is acidified to a pH of 4.0-5.5, the fluid is diluted, and the diluted, acidified fluid is subjected to cross-flow diafiltration in a filtration unit with a mean pore size of 0.1-1.2 micrometers. The volume of the fluid is kept constant throughout this initial filtration by adding sodium-chloride solution. This initial diafiltration retains casein, and the permeate is a clear solution that contains all the whey proteins and low molecular weight components. The low molecular weight components are then removed from the first permeate by means of a second cross-flow diafiltration in a filtration unit with a mean pore size of 5,000-80,000 daltons, preferably 10,000 daltons. The pH of the fluid for this second filtration is kept between 4.0-5.5. The retentate of the second cross-flow diafiltration constitutes the immunoglobulin product sought. In such a process, immunoglobulins are reported to be harvested from colostrum or hyperimmunized milk, but this result is reported to be achieved by means of easily fouled hollow fiber membranes and the immunoglobulins were not enriched compared to the other protein species. An attempt is made to overcome the handicap of hollow fiber membranes by extensive use of diafiltration which renders this process not practical for large industrial applications.
Japanese patent publication 60-248152 relates to a method of manufacturing calcium fortified salts and their use. In the disclosed method, concentrated, acidified (pH adjusted to 5.0 or below) cheese whey is filtered through such membranes as an ultrafiltration membrane and an electrodialytic membrane to allow calcium to come out through the membrane in its ionic state in solution with phosphate and citrate ions. The filtrate thus obtained is adjusted to pH 6.0-9.0 by the addition of alkaline solution so as to obtain a precipitate of calcium phosphate and calcium citrate which is isolated by centrifugation. This patent does not disclose a means by which an enrichment and concentration of proteins in a fluid can be obtained.
Thomas et al., U.S. Pat. No. 4,716,044 relates to an improved process for obtaining juice from fruit. The disclosed process subjects a fluid puree of fruit and juice to a single ultrafiltration through a rigid porous tubular housing which has a food grade ultrafiltration membrane, preferably a metallic oxide membrane, deposited on its inside surfaces. During the single pass, water soluble sugars, organic acids, flavor compounds and the like reportedly pass through the disclosed membrane while insoluble solids, proteins of molecular weight greater than about 14,000, and all microorganisms reportedly are retained in the fruit retentate. Although this patent does disclose a means by which a concentration of proteins in a fluid occurs, it does not contemplate enriching a specific protein fraction of a fluid by means of ultrafiltration and a pH shift.
Trulson et al., U.S. Pat. No. 3,977,967 relates to an ultrafiltration apparatus to be used for the concentration and separaton of components contained in liquids. The disclosed apparatus is reportedly comprised of a module which contains a plurality of axially aligned, hollow tubular members which have a defined porosity and a substantially uniform, continuous, adherent, porous coating of preformed, aggregated, inorganic metal oxide particles on their interior or exterior surface. In the disclosed process, low molecular weight dissolved phases reportedly permeate the walls of the tubes while the larger diameter molecules are retained in the liquid. In a preferred embodiment of the disclosure, the metal oxide coated onto the ultrafiltration apparatus is zirconia. Trulson et al. report that this ultrafiltration apparatus can be used to concentrate and separate the protein fraction of cottage cheese whey from the bulk of the water, lactose and dissolved salts. They do not indicate, however, that the constituent proteins of that protein fraction can be concentrated and separated by their apparatus, or that such a separation and concentration can be achieved by altering the pH at which the fluid is subjected to ultrafiltration.
Johnson et al., U.S. Pat. No. 3,431,201 relates to an improved hyperfiltration process for reducing the concentration of solute in an aqueous solution. The improvement comprises contacting an aqueous feed solution with a hydrous metal oxide ion exchange mass so that the ions which are deleterious to hydrous metal oxide membranes may be removed from the feed solution prior to hyperfiltration through that form of membrane. By removing the interfering ions, this process reportedly increases the useful life of the membrane, provides a method for controlling the pH of the feed solution, and provides a method for adding membrane-forming material to the membrane. Johnson et al. report that solute rejection by a hydrous zirconium oxide membrane during hyperfiltration can be increased by modifying the pH of the feed solution to increase the anion exchange properties of the dynamic membrane. Johnson et al. modify pH to separate molecules on the basis of charge. They do not contemplate, however, separating components on the basis of size, where the effective "permeability size" of a component is controlled by modifying the pH of the fluid.